This invention is concerned with novel apparatus, materials and methodologies for synthesizing chemical compounds, especially oligomers. Chemical reactions are accomplished on surfaces in a fashion which is both easy and economical and which is amenable to the attainment of high yields. Automation of chemical reaction processes is facilitated in the present invention. Synthesis of oligomers, especially oligonucleotides and polypeptides, is especially benefitted by the employment of the present invention. A wide variety of other chemical reactions can be achieved in accordance with the present invention, however. The present invention is also suited for the preparation of chemical libraries which are useful per se, inter alia for screening purposes and otherwise.
It has been proposed heretofore to employ apparatus commonly called an xe2x80x9cink jetxe2x80x9d for the delivery of deblocking reagents in solid state oligomeric reactions. Such proposal, however, is crude, is limited in scope, and generally requires non-automatable procedures for its employment. Thus, it has been proposed to use an xe2x80x9cink jetxe2x80x9d apparatus to place droplets of a deblocking reagent, zinc bromide, upon specified locations of a reaction surface to deblock a growing oligonucleotide chain to render it amenable to chain elongation. This has been proposed for use, for example, on a microscope slide. Following the delivery of deblocking reagent, it was proposed to manipulate the microscope slide such as by dipping it into a quantity of further reagent to accomplish a chain elongation. Further application of the xe2x80x9cink jetxe2x80x9d delivery of chemical reagent was then proposed, however realignment of the microscope slide would be necessitated by that proposed methodology. In addition to the cumbersome nature of the prior proposal and its lack of suitability to full automation, only relatively small harvests of oligomeric product were anticipated using the proposed scheme.
There is a great need for chemical reaction apparatus, materials and attendant methodologies which permit the automated, high yield, relatively large scale synthesis of chemical species, especially oligomers. The apparatus and methodologies provided by the present invention now enable the use of chemical xe2x80x9cjettingxe2x80x9d technology for the practical synthesis of chemical and biochemical products in high yield and with ease of synthesis. There is also provided a need for synthetic systems which deliver complex synthesized products to receiving vessels with ease and in high yield.
The apparatus and methods of the present invention also address a long-felt need by permitting the preparation of libraries of chemical compounds having predictable diversity among the functional moieties thereupon.
This invention also diminishes waste stream pollution associate with many prior synthetic technologies through the precision application of reagent moieties in synthetic schemes.
Precisely arrayed pluralities of defined chemical compounds are also possible through employment of this invention. Binding, reaction, degradation, chemical and biological interaction and other testing protocols may, thus, be performed with unparalleled convenience through practice of embodiments of this invention.
The invention minimizes reagent usage such that the impact of toxic, explosive, radioactive, or expensive sensitive materials on syntheses is reduced.
In accordance with the present invention, there are provided chemical reaction apparatuses. These apparatuses comprise a reaction support having a first surface (alternatively called a reaction surface) thereupon. The reaction surface is considered to have a plurality of preselected reaction sites upon it. These are generally in a regular geometric array such as a grid, but may be in other patterns as well. The apparatus further comprises a first droplet generator for jetting reactant droplets upon the first surface. The apparatus further comprises a second droplet generator for jetting droplets of a second reactant species upon the first surface. The droplets of each of the first and second droplet generators are under control of a control means such as a general or special purpose digital computer, with attendant switches and actuators, for causing the droplets from each of the droplet generators to impact upon definable sets of the preselected reaction sites on the reaction surface of the reaction support. The impacting of the droplets upon the sets of reaction sites is preferably in accordance with a preselected pattern or patterns. In this way, chemical reactants can be directed to particular reaction sites upon the reaction surface in any desired order so as to achieve desired chemical reactions at such reaction site without the need for removing, manipulating, and redeploying the reaction support.
In accordance with preferred embodiments, three, four, or more droplet generators are provided, each connected to one or more chemical reactant reservoirs, in order to provide diversity among the chemical reactions possible at the reaction surface. The control means is preferably programmed to deliver different sequences of reactants at different reaction sites so as to synthesize differing chemical compounds at such sites. Preferably, the methods of the invention are performed iteratively such that relative complex molecules, such as oligomers, can be synthesized.
Synthesis has been found to be benefitted by employment of porous reaction supports, especially those having the capability of transporting fluid from the first (or reaction) surface to a second surface thereof. Improvement of yield through exploitation of the internal pore volume of the support and amenability for improved product harvest is also now possible.
Once the different chemical moieties have been synthesized at the plurality of reaction sites, they may be employed in a variety of highly useful ways. Thus, the different chemical moieties may be exposed to test solutions, such as bodily fluids of a test animal, to diagnose or ascertain bodily states in such animal. A wide variety of assays may thus be formulated using the apparatus and methodologies of the present invention. In accordance with other utilities, the chemical species thus formed may be used as probes in biological systems or as primers or substrates for polymerase chain reaction amplification or the like. Hybridization studies may also be conducted using apparatus and methods in accordance with the present invention, where the products of the methodologies are oligonucleotides, polypeptides or other hybridizable species. All of the foregoing utilities are known per se to those skilled in the art.
In accordance with other preferred embodiments of the invention, reaction apparatuses are provided where one or more droplet generators, each in fluid communication with pluralities of reactant reservoirs, are employed to perform synthesis. Control means are caused to effect the operation of valving moieties to select reactants in appropriate orders to achieve the desired reactions at individual reaction sites.
In accordance with the invention, methods for synthesizing chemical species comprise identifying a plurality of reaction sites upon a reaction surface and jetting upon a first set of such reaction sites, droplets of fluid comprising a first chemical reactant. The methods also comprise jetting upon a second set of such reaction sites, droplets of fluid comprising a second chemical reactant species. It will be appreciated that, through practice of the present invention, the first jetted reactant species and the second jetted reactant species may be jetted to the same or different reaction sites on the reaction surface Control means for effectuating the jetting of such fluids upon the selected sets of reaction sites is also invoked to attain the ends of the invention. Iterative jetting of reactant species permits the elaboration of wide varieties of chemical moieties as the reaction sites and permits the generation of libraries of diverse species.
It is also possible to employ the present invention in a hybrid fashion by combining it with other chemical reaction schemes. Thus, for example, a support for reaction may be coated with an initial reactant species and then reacted through the jetting of chemical reactants in preselected locations on the surface. Subsequent reaction at the selected locations on the surface in accordance with the invention may ensue whereby a plurality of reactant species are delivered to such locations. Thus, by pretreating the support where the reaction is to occur with a first reactant moiety, certain economies of scale may be attained. The employment of chemical jetting technology to deliver often expensive reagents for at least some of the subsequent steps, is, however, preferred.
At any reaction site, either or both of the first and second reactants may be delivered thereto. Thus, at the reaction site, either the first reactant, the second reactant, or the first reactant followed by the second reactant can be so delivered. The ensuing chemical reactions will depend upon the identity and order of chemical reactants delivered to any particular reactant site.
This is also true where an initial reaction species is applied to the support prior to the delivery of jetted reactants. In such case, at reaction site Rn, one of four situations will prevail after jetting of two reactants has occurred. First, it may be that neither the first nor the second reactant are directed to a given reaction site. In this case, only the initial reaction species will have been delivered to this site. In two additional cases, either the first reactant or the second reactant, but not both is jetted to site. In this case, two different combinations of two reagents are delivered to the site. Finally, it may be seen that both the first and the second reactant can be jetted to the site whereupon three reaction species will have been so delivered. Persons of ordinary skill in the art will readily appreciate how a complex series of reactants can be delivered to particular sites on a reaction surface to achieve complex and varied chemical reactions. The present invention is suited to the delivery of a large variety of chemical species including reagents, intermediates, blocking and deblocking agents, monomers, dimers, oligomers, solvents, washing agents, cleaving agents and the like.
In accordance with preferred embodiments, the methodologies of the present invention are performed iteratively. Thus, three, four, five, and more reactants can be delivered to a reaction surface in varying combinations at different reaction sites on the surface. The number of different chemical moieties which may, thus, be elaborated is extraordinarily numerous and varied. It is, thus, possible to generate, isolate and recover a wide variety of different chemical species in a highly automated fashion on small reaction surfaces. The present invention also provides reaction assemblies wherein a reaction support surmounts a collection plate, preferably one having a plurality of collection wells. Transport of chemical species through the reaction support enables their collection in the collection wells where they may be easily recovered, analyzed, tested, hybridized, screened, assayed and otherwise utilized. The efficient deposition of product species into such collection vessels is a particularly advantageous aspect of the invention.